Rhodopsin kinase activity in the mammalian pineal gland and other tissues.

Rhodopsin kinase, an enzyme involved in photochemical transduction in the retina, has been found in the mammalian pineal gland in amounts equal to those in the retina; other tissues had 7 percent of this amount, or less. This finding suggests that, in mammals, rhodopsin kinase functions in the pineal gland and other tissues to phosphorylate rhodopsin-like integral membrane receptors and is thereby involved in signal transduction.

Melatonin synthesis: adenosine 3',5'-monophosphate and norepinephrine stimulate N-acetyltransferase.

Treatment of cultured rat pineal glands with norepinephrine or dibutyryl adenosine 3',5'-monophosphate causes a six- to tenfold stimulation of N-acetyltransferase. This enzyme converts serotonin to N-acetylserotonin, the immediate precursor of melatonin. The increased synthesis of melatonin caused by norepinephrine treatment appears to be the result of stimulation of N-acetyltransferase by an adenosine 3',5'-monophosphate mechanism.

Indole metabolism in the pineal gland: a circadian rhythm in N-acetyltransferase.

The activity of N-acetyltransferase in the rat pineal gland is more than 15 times higher at night than during the day. This circadian rhythm persists in complete darkness, or in blinded animals, and is suppressed in constant lighting. The N-acetyltransferase rhythm is 180 degrees out of phase with the serotonin rhythm and is similar to the norepinephrine and melatonin rhythms. Experiments in vitro indicate that norepinephrine, not serotonin, regulates the activity of N-acetyl-transferase through a highly specific receptor.

Melatonin inhibition of the neonatal pituitary response to luteinizing hormone-releasing factor.

Neonatal rat anterior pituitary glands treated in organ culture with 1 nanomolar luteinzing hormone-releasing factor (LRF) showed a tenfold increase in medium luteinizing hormone (LH) concentrations over control values. Simultaneous treatment of the glands with 1 nanomolar melatonin significantly reduced the stimulatory effect of LRF on release of LH. This finding indicates that melatonin can act directly on the neonatal pituitary to inhibit the LH response to LRF.

Rapid light-induced decrease in pineal serotonin N-acetyltransferase activity.

Light acting by way of the eye causes the dark-induced activity of serotonin N-acetyltransferase in the pineal gland of the rat to decrease with a halving time of about 3 minutes. This effect, which is one of the more rapid physiological changes known to occur in the activity of any enzyme that metabolizes biogenic amines, appears to explain the rapid increase in the concentration of pineal serotonin that is caused by light exposure at night.

Biosynthesis of biopterin: adrenergic cyclic adenosine monophosphate-dependent inhibition in the pineal gland.

Pineal glands in organ culture synthesize and release biopterin and are able to maintain concentrations of biopterin occurring in vivo for up to 54 hours in vitro. The intracellular biopterin content is reduced 50 percent by treatment with l-norepinephrine or cyclic adenosine monophosphate derivatives, but not by d-norepinephrine. This is an indication that biopterin levels are regulated by an adrenergic cyclic adenosine monophosphate-dependent mechanism. The decline in tissue biopterin content, produced mainly by inhibited of biosynthesis, is maximal at 6 hours and is not associated with either an increase in biopterin release or a shift in the reduction state of the biopterin.

Pineal N-acetyltransferase is inactivated by disulfide-containing peptides: insulin is the most potent.

Pineal N-acetyltransferase can be inactivated in broken cell preparations by cystamine through a mechanism of thiol-disulfide exchange. Some, but not all, disulfide-containing peptides can inactivate this enzyme; the most potent inactivator is insulin. These findings suggest that a disulfide-containing peptide with high reactivity toward N-acetyltransferase may participate in the intracellular regulation of this enzyme.

Arginine vasotocin: effects on development of reproductive organs.

Immature 25-day-old mice were injected daily with 1 microgram of arginine vasotocin for 3 or 4 days and killed 24 hours after the last injection. The ovaries were 30 percent smaller in treated females than in controls. The ventral prostates and accessory organs (seminal vesicles and coagulating glands) were less than half the size of these structures in control males. Similar results were observed when 15-day-old mice were given similar injections and killed 2 weeks after the last injection; furthermore, testis weights were 28 percent smaller than those of controls. It is speculated that arginine vasotocin, which has been found in mammalian pineal glands, might mediate effects of the pineal gland on normal sexual development.

Pineal gland: dibutyryl cyclic adenosine monophosphate stimulation of labeled melatonin production.

In organ cultures of intact rat pineal glands, N(6)O(2')-dibutyryl adenosine 3', 5'-monophosphate stimulates the conversion of tritiated trytophan to tritiated melatonin, as does L-norepinephrine. Potential sites of stimulation of melatonin production by dibutyryl cyclic adenosine monophosphate are discussed, based on observations that the dibutyryl analog also stimulates the conversion of serotonin labeled with carbon-14 to carbon-14-labeled melatonin without altering hydroxyin-dole-O-methyl transferase activity or intracellular accumulation of serotonin labeled with carbon-14.

Pineal serotonin N-acetyltransferase activity: abrupt decrease in adenosine 3',5'-monophosphate may be signal for "turnoff".

Dispersed pinealocytes have been used to study the role of adenosine 3',5'-monophosphate (cyclic AMP) in the "turnoff" of N-acetyltransferace activity. Activity was first stimulated 100-fold by treating cells with 1-norepinephrine. 1-Propranolol acted stereospecifically to rapidly reverse this, resulting in a 70 percent loss of enzyme activity within 15 minutes. An even more rapid 1-propranolol-induced decreased in cyclic AMP also occurred. This together with the observation that the inhibitory effect of 1-propranolol on N-acetyltransferase was blocked by dibutyryl cyclic AMP and phosphodiesterase inhibitors indicate that an abrupt decrease in cyclic AMP may be the signal for the rapid decrease in pineal N-acetyltransferase activity.

5-hydroxytryptophan elevates serum melatonin.

Daytime administration of 5-hydroxytryptophan to sheep elevated serum melatonin more than sevenfold within 2 hours. This suggests that administration of 5-hydroxytryptophan could be used as the basis of a clinical test of pineal function and that melatonin might mediate some clinical effects of 5-hydroxytryptophan.

Pineal serotonin N-acetyltransferase: expression cloning and molecular analysis.

Pineal serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, or AA-NAT) generates the large circadian rhythm in melatonin, the hormone that coordinates daily and seasonal physiology in some mammals. Complementary DNA encoding ovine AA-NAT was cloned. The abundance of AA-NAT messenger RNA (mRNA) during the day was high in the ovine pineal gland and somewhat lower in retina. AA-NAT mRNA was found unexpectedly in the pituitary gland and in some brain regions. The night-to-day ratio of ovine pineal AA-NAT mRNA is less than 2. In contrast, the ratio exceeds 150 in rats. AA-NAT represents a family within a large superfamily of acetyltransferases.

Pinealocyte projections into the mammalian brain revealed with S-antigen antiserum.

Neural processes from mammalian pinealocytes have been discovered in several brain areas. These processes were visualized immunocytochemically in the Djungarian hamster, Phodopus sungorus, with an antiserum against bovine retinal S-antigen and traced as far as the region of the posterior commissure and habenular nuclei. This result indicates that pineal-to-brain connections exist in the mammal, and that the mammalian pineal gland, currently thought of only as a neuroendocrine organ, may communicate directly with select brain regions by way of these projections. The existence of mammalian pinealocyte projections is consistent with the view that these cells are not of glial origin but are derivatives of photoreceptor cells of the pineal complex of lower vertebrates that transmit signals to the brain by neural projections.

Melatonin production: proteasomal proteolysis in serotonin N-acetyltransferase regulation.

The nocturnal increase in circulating melatonin in vertebrates is regulated by 10- to 100-fold increases in pineal serotonin N-acetyltransferase (AA-NAT) activity. Changes in the amount of AA-NAT protein were shown to parallel changes in AA-NAT activity. When neural stimulation was switched off by either light exposure or L-propranolol-induced beta-adrenergic blockade, both AA-NAT activity and protein decreased rapidly. Effects of L-propranolol were blocked in vitro by dibutyryl adenosine 3',5'-monophosphate (cAMP) or inhibitors of proteasomal proteolysis. This result indicates that adrenergic-cAMP regulation of AA-NAT is mediated by rapid reversible control of selective proteasomal proteolysis. Similar proteasome-based mechanisms may function widely as selective molecular switches in vertebrate neural systems.

Transcriptional regulation of arylalkylamine-N-acetyltransferase-2 gene in the pineal gland of the gilthead seabream.

Pineal serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light-entrainable clock-containing PAC-2 zebrafish cell line, a stably transfected seabream aanat2 promoter-luciferase DNA construct exhibited a clock-controlled circadian rhythm of luciferase activity, characteristic for an E-box-driven expression. In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms.

New light is shining on the melatonin rhythm enzyme: the first postcloning view.

One of the most interesting molecules in circadian biology is serotonin N-acetyltransferase (arylalkyfamine N-acetyltransferase, AANAT), the enzyme that controls the daily rhythm in pineal melatonin production and blood melatonin. The recent cloning of AANAT cDNA has led to the characterization of the human gene; the realization that AANAT represents a unique gene family; the discovery of circadian rhythms in AANAT mRNA; the determination of the basis of transsynaptic and cellular regulation of expression of the AANAT gene; a new understanding of the relationship of AANAT mRNA and activity; and the surprising finding of strong expression of the AANAT gene in the retina and significant levels in select brain regions, the pituitary gland, and testes. The cloning of AANAT cDNA has not only made it possible to answer longstanding questions in circadian biology, but has also raised stimulating new issues.

Adenosine stimulates adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate accumulation in rat pinealocytes: evidence for a role for adenosine in pineal neurotransmission.

Adenosine produces a concentration-dependent increase in pinealocyte cAMP (EC50, approximately 0.3 nM) and cGMP accumulation (EC50, approximately 0.7 nM). Maximal increases in both nucleotides are evident 10 min after treatment; 1 h later values return to pretreatment levels. Concentration-dependent effects on cAMP are also observed with N6-(L-2-phenylisopropyl)adenosine (EC50, approximately 0.75 nM), 5'-N-ethylcarboxy aminoadenosine (EC50, approximately 0.75 nM), and 2-chloroadenosine (EC50, approximately 2.0 nM); the EC50 values for stimulation of cGMP with these agents are higher by a factor of 2-10. In the case of 5'-N-ethylcarboxy amidoadenosine, the concentration-response curve is biphasic, with a significant effect evident within the range of 1-100 pM. The stimulatory nature of this response and the relative potency of the agonists tested are consistent with the involvement of an A2-like adenosine receptor. Comparison of adenosine and the selective beta-adrenergic agonist isoproterenol indicated that their maximal EC50 values were generally similar. Studies with antagonists revealed that both 8-(p-sulfophenyl)theophylline (1 microM) and the xanthine amine congener (8-[4-[[[(2-aminoethyl)carbonyl]methyl]oxy]phenyl]1,3- dipropylxanthine (1 microM) inhibited the effects of adenosine (1 nM to 1 microM), but xanthine amine congener was more potent; the latter was markedly effective at 0.1 nM, whereas 8-(p-sulfophenyl)theophylline was nearly ineffective at this concentration. It was also determined that pineal cells generate extracellular adenosine from extracellular ATP. ATP is thought to be released along with catecholamines during neurotransmission. Hence, these studies support the view that adenosine could participate in the transsynaptic regulation of pineal function.

Melatonin inhibits gonadotropin-releasing hormone-induced elevation of intracellular Ca2+ in neonatal rat pituitary cells.

GnRH stimulates LH release by increasing intracellular Ca2+ ([Ca2+]i). Melatonin is known to inhibit GnRH-stimulated LH release from neonatal rat pituitary cells. In the present report, the issue of whether melatonin acts through [Ca2+]i was addressed. [Ca2+]i was studied in cells in suspension, using Fluo-3 as a fluorescent indicator. In neonatal rat pituitary cells, melatonin inhibited the GnRH-induced [Ca2+]i increase in a dose-dependent manner; the GnRH-induced increase in [Ca2+]i was inhibited 40% by 100 nM melatonin. The relative potencies of several indoles as inhibitors of the GnRH stimulation of [Ca2+]i in neonatal pituitary cells (2-iodo-melatonin greater than melatonin greater than 6-hydroxymelatonin) correlate with their known potencies to inhibit LH release and with their binding affinity to high affinity melatonin receptors, which indicates that these receptors probably mediate the effects of melatonin. Further support for this interpretation comes from the observation that melatonin does not inhibit the GnRH effect on [Ca2+]i in cells obtained from adolescent rat pituitary glands, which lack melatonin receptors and are insensitive to melatonin as an inhibitor of GnRH-stimulated LH release. The possible involvement of an inhibitory G-protein was also investigated by studying the effects of pertussis toxin. Pretreatment with pertussis toxin antagonized the effects of melatonin on [Ca2+]i and LH release. This indicates that melatonin may inhibit the GnRH-induced increase in [Ca2+]i through a mechanism involving a pertussis toxin-sensitive G-protein. To examine the role of extracellular Ca2+ in this effect, the effects of melatonin were examined in a low Ca2+ medium. Under these conditions, the effect of melatonin was markedly reduced, which indicates that melatonin may act by inhibiting Ca2+ influx. These observations indicate that melatonin inhibits GnRH stimulation of [Ca2+]i in neonatal rat gonadotrophs, and this probably explains the inhibitory action of melatonin on GnRH stimulation of LH release.

Transport of maternal[3H]melatonin to suckling rats and the fate of [3H]melatonin in the neonatal rat.

The question of whether maternal melatonin could be transported in milk to suckling rats was investigated because melatonin is probably not produced in these animals during the first 10 days of life. [3H]Melatonin was found to be rapidly transferred from the maternal circulation into lactating mammary tissue, and the stomach of each suckling rat was found to contain [3H]melatonin. To study the fate and tissue distribution of [3H]melatonin originating in the neonatal stomach, suckling rats were given [3H]melatonin by stomach tube; [3H]melatonin was recovered from plasma and seven tissues, including brain, 15 and 60 min later. The general tissue distribution of [3H]melatonin was similar to that found in adult rats. The major [3H]melatonin metabolites in the urine of suckling rats, as the adult rats, were the conjugates of 6-hydroxy-melatonin.

A suspension culture of pinealocytes: regulation of N-acetyltransferase activity.

A method to prepare suspension cultures of highly viable pinealocytes is described. Pineal glands of neonatal or adult rats can be used; the best yield is obtained from neonatal glands. Morphological examination of the cells indicates that they have a normal, healthy appearance and retain some structures typical of neonatal pinealocytes. During the first 24 h of culture, the cells aggregate into small clusters; after several days, larger aggregates can be seen. Biochemical studies indicate that adrenergic stimulation of these cells causes a 30- to 100-fold increase in serotonin N-acetyltransferase activity, a response which is unique to the pineal gland. The relative order of potency of several adrenergic agonists is l-isoproterenol greater than l-norepinephrine greater than or equal to l-epinephrine greater than phenylephrine. Serotonin, tyramine, histamine, carbachol, gamma-aminobutyric acid, cocaine, and desmethylimipramine are inactive. Studies using adrenergic and metabolic inhibitors indicate that the regulation of N-acetyltransferase in these cells has the same characteristics of that seen in adult tissue. In addition, these cells synthesize protein and RNA from radioactive precursors, convert tryptophan to serotonin, and N-acetylate endogenous serotonin.